FIG 5.
Mixtures of HIV-infected and uninfected T cells, treated with proteasome inhibitors, select for the preferential survival of uninfected cells. (A) Jurkat T cells and chronically HIV-infected J-Lat 10.6 cells were treated with apoptosis-inducing anti-Fas (CH11), and cell survival was monitored by intracellular ATP. Depicted are means (±SD) of the results of triplicate experiments. (B) Jurkat T cells and J-Lat 10.6 cells were treated with camptothecin, and cell survival was monitored by intracellular ATP content. Depicted are means (±SD) of the results of triplicate experiments. (C) J-Lat 10.6 cells were labeled with the lipophilic dye CTO to distinguish them from unlabeled Jurkat T cells, and the two cell types were mixed at a 1:1 ratio. T20 was added to prevent spreading HIV infection, and then the cells were treated with control, bortezomib, or ixazomib and analyzed for active caspase 3 staining. Depicted is the proportion of live (active caspase 3-negative) cells that were also CTO-positive (J-Lat 10.6) cells (means ± SD of the results of three independent experiments). (D) Pooled data from three replicates of the experiment shown in panel C depicting means and SD. (E) Representative flow data from panels C and D; cell death in the CTO-positive (J-Lat) and CTO-negative (Jurkat) cells can be seen in the same sample. (F) Primary activated CD4 T cells were mock infected or infected with HIV-1RF, treated with ixazomib (100 nM) or vehicle control, and assessed for active caspase 3 expression over time. (G) Freshly isolated CD4+ T cells from HIV-infected ART-suppressed patients were treated with ixazomib or control and ART (T20, EFV, or RAL) to prevent spreading infection. After 96 h, cells were harvested, both total (top) (n = 11 patients) and integrated (bottom) (n = 8 patients) HIV DNA copies per million cells were determined, and the values were compared to the control by one-sample t test. *, P < 0.05; **, P < 0.01; ***, P < 0.001.